Cystic fibrosis is a common genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl-channel with novel properties. Knowledge of the structure and function of CFTR remains superficial. The main goal of this research is to better understand how CFTR forms a Cl-channel with a specific focus on the membrane spanning domains (MSDs) and their interaction with the rest of the protein. Specific Aim 1 focuses on understanding how the MSDs contribute to the formation of the channel pore by addressing several questions. 1) How do different amino acid sequences in Xenopus and human CFTR generate differences in function? The development and study of chimeric proteins will identify regions and specific residues that confer the unique properties associated with CFTR. 2) Do proline residues in the MSDs contribute to the formation of the channel pore? Proline has unique properties that are predicted to confer a bend in the helices forming the channel. By studying the effect of replacing these prolines an assessment of channel structure will be obtained. 3) Which residues line the channel pore? Blockers that irreversibly interact with introduced cysteines will identify residues that line the pore. 4) How do membrane- spanning sequences associate to form the channel? The studies will use a biochemical approach to test the hypothesis that transmembrane sequences in CFTR contain explicit structural information that causes them to form specific interactions. Specific Aim 2 focuses on the role that the intracellular loops (ICLs) play in controlling function and in protein:protein interactions. 1) Do intracellular loops determine the conduction or regulatory properties of CFTR Cl-channels? The patch-clamp technique will be used to test how changes in the ICLs affect channel function with particular emphasis given to ICLs that contain clusters of CF mutations. 2) Do intracellular loops interact with cytosolic domains? The work will test the hypothesis that direct physical interactions exist between the ICLs and the cytoplasmic regulatory domains. The results will provide new insights into the structure and function of CFTR and the way that CF-associated mutations disrupt function. The results will also increase understanding of the structure of other medically important ABC transporters and may give new insight into the construction of ion channels.